SARS-CoV S、N、E和M DNA免疫的细胞免疫应答

发布时间：2018-02-28 08:11

本文关键词： 冠状病毒 SARS DNA免疫细胞因子　出处：《新疆农业大学》2006年硕士论文　论文类型：学位论文

【摘要】： SARS ( Severe Acute Respiratory Syndrome)是一种传染性强、病死率高、严重危害人民生命健康的传染病。世界卫生组织于2003年4月6日宣布引起SARS的病原体是一种新的病毒,属于冠状病毒家族,称为SARS相关冠状病毒(SARS-Associated coronavirus, SARS-CoV )。SARS-CoV表现出许多不同于冠状病毒的特性,为冠状病毒中一个新的分支。目前,病毒性疾病尚无特效治疗方法,对于SARS也是一样。所以,控制SARS的最好办法是预防,而疫苗的开发则是建立有效防范措施的关键环节。DNA疫苗是继灭活疫苗、亚单位疫苗之后的第三代疫苗,具有许多的优越性。所以本研究选择了SARS病毒DNA疫苗这一方向,以SARS-CoV主要结构蛋白S、N、E和M蛋白基因作为研究对象。构建针对SARS-CoV结构蛋白基因的重组核酸疫苗,观察其免疫小鼠后机体的特异性免疫应答的情况,探讨其作为SARS-CoV疫苗的可能性。本研究由两部分组成: 第一部分为SARS-CoV结构蛋白基因重组核酸疫苗的构建。从GenBank上获得SARS(BJ01)S、N、E和M蛋白基因的序列,使用引物设计软件Primer6.0进行引物设计。以SARS-CoV(BJ01)cDNA为模板,合适条件下进行PCR,获得SARS-CoV SARS-CoV S、N、E和M蛋白基因片段。经酶切处理后,定向克隆于pVAX1真核表达载体,构建包含SARS-CoV S、N、E和M基因片段的重组质粒pVAX/S、pVAX/N、pVAX/E和pVAX/M。经酶切和测序等方法鉴定重组质粒的正确性。然后利用间接免疫荧光法鉴定重组质粒的体外表达。将重组质粒瞬时转染真核细胞。48小时后IFA试验检测目的基因的表达产物的活性。第二部分为真核表达质粒pVAX/S、pVAX/N、pVAX/E和pVAX/M在小鼠体内的免疫原性研究。将重组质粒按100ug/只肌肉注射BALB/c小鼠,第一次免疫后2周进行加强免疫,4周后进行第三次免疫。试验分为空载体组,实验组。在免疫后的2、4、6、8、10周分别采集并分离小鼠血清。用流式细胞仪检测相关细胞因子TNF、IFN-?、IL-5、IL-4、Il-2水平。实验结果显示,重组质粒DNA免疫小鼠后能够诱导机体产生特异的细胞免疫和体液免疫应答。这为进一步研制安全、有效的SARS DNA疫苗奠定了基础。
[Abstract]:SARS (Severe Acute Respiratory Syndromeis) is a kind of infectious disease with high mortality and serious harm to people's life and health. In April 6th 2003, the World Health Organization declared that the pathogen causing SARS is a new virus, belonging to the coronavirus family. Called SARS associated coronavirus. SARS-CoV coronavirus.SARS-CoV shows many characteristics different from coronavirus.It is a new branch of coronavirus. At present, there is no special treatment for viral diseases, and the same is true for SARS. The best way to control SARS is to prevent it, and the development of vaccine is the key link to establish effective preventive measures. DNA vaccine is the third generation vaccine after inactivated vaccine and subunit vaccine. Therefore, we chose the direction of DNA vaccine of SARS virus to construct recombinant nucleic acid vaccine against SARS-CoV structural protein gene. To observe the specific immune response of mice immunized with it and to explore the possibility of its use as SARS-CoV vaccine. This study consists of two parts:. The first part is the construction of recombinant nucleic acid vaccine for structural protein gene of SARS-CoV. The sequence of SARS-CoV BJ01SJ01SN and M protein gene was obtained from GenBank. Primer design software Primer6.0 was used for primer design. SARS-CoV(BJ01)cDNA was used as template. Under suitable conditions, SARS-CoV SARS-CoV signon E and M protein fragments were obtained and cloned into pVAX1 eukaryotic expression vector after restriction endonuclease digestion. The recombinant plasmids pVAX / SapVAX / pVAXP / NV AXR / E and pVAXR / M were constructed. The correctness of the recombinant plasmids was confirmed by restriction endonuclease digestion and sequencing. The in vitro expression of the recombinant plasmids was identified by indirect immunofluorescence assay. The transient expression of the recombinant plasmids was determined. The activity of the expressed products of the target gene was detected by IFA assay after transfection of eukaryotic cells. In the second part, the immunogenicity of the eukaryotic expression plasmid pVAX- pVAX / NV AX / NV AXP / E and pVAX/M in mice was studied. The recombinant plasmid was injected intramuscularly into BALB/c mice. The experiment was divided into empty carrier group and experimental group. Serum samples were collected and isolated from mice at 10 weeks after immunization. TNFF IFN-IFN was detected by flow cytometry. IL-5, IL-4 and Il-2 levels. The results showed that the recombinant plasmid DNA could induce specific cellular and humoral immune responses in mice, which laid a foundation for the further development of a safe and effective SARS DNA vaccine.
【学位授予单位】：新疆农业大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R392

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